An individual yeast cell can divide only a certain number of times; however, the daughter produced during budding has the same probability of a full replicative life span as its mother had at the beginning of hers. A handful of genes that determine the life span of the yeast cell has been identified and cloned. The overall goals of this proposal are to elucidate the function of these genes in determining life span, and to identify the molecular basis for the age-dependent expression of these genes. The specific aims are as follows; (1) The interactions of available yeast longevity-assurance genes (LAGs) will be examined to provide an estimate of the number of different pathways that are involved in determining yeast replicative life span and as a first step in fleshing out these pathways genetically. (2) New mutants in LAG1 and LAG2 will be made by in vitro mutagenesis to aid in the identification of interacting genes and in the delineation of the functional domains of Lag1 and Lag2. (3) Additional genes that interact with yeast LAGs will be uncovered and analyzed by screening for gene dosage (multicopy) suppressors, extragenic (second site) suppressors, and mutants that display synthetic phenotypes with selected LAG mutants. The genes that will be identified in these ways will be characterized. (4) Transcriptional control of LAG1 and LAG2 and mRNA stability, during the yeast life span, will be examined. The intracellular localization of the proteins will be determined in young and old cells, as well as the abundance of the proteins and their phosphorylation status. These studies will serve to define the levels at which the expression and activity of the LAGs are regulated. (5) The LAG1 promoter will be subjected to deletion and to footprint analysis. Proteins that interact with functional promoter regions will be assayed by gel mobility-shift, as a prelude to the cloning of the cognate genes. (6) The longevity-assurance genes studied in detail thus far are preferentially expressed in young yeast cells. The genes that appear to be preferentially expressed in old yeasts will now be characterized more fully in terms of their impact on longevity. The goal is to address the issue of whether active life span- determining processes are at work in old yeasts. All of these studies should provide a better understanding of the molecular and cellular events associated with aging. There are reasons to believe that there may exist some commonality between these events in yeasts and in humans. Thus, an elucidation of the function and regulation of this and other yeast longevity-assurance genes may contribute to interventions that may improve the health and function of older humans.